title: Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys Vitamin D Total protein binding assay.
abstract: BACKGROUND
  Presence of the 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] metabolite affects accurate determination of 25(OH)D3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys).
  
  
  METHODS
  Neonatal samples, containing up to 58% of 3-epi-25(OH)D3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D3 and 3-epi-25(OH)D3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D3.
  
  
  RESULTS
  The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D3 recognition by the CPB assay when present as the natural endogenous metabolite.
  
  
  CONCLUSIONS
  The automated CPB assay displays minimal 3-epi-25(OH)D3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D3. Exogenous 3-epi-25(OH)D3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno- or protein binding assays.
authors.forename: Johannes M W
authors.initials: JM
authors.lastname: van den Ouweland
authors.forename: Antonius M
authors.initials: AM
authors.lastname: Beijers
authors.forename: Henny
authors.initials: H
authors.lastname: van Daal
authors.forename: Marc G L M
authors.initials: MG
authors.lastname: Elisen
authors.forename: Gerard
authors.initials: G
authors.lastname: Steen
authors.forename: Jos P M
authors.initials: JP
authors.lastname: Wielders
mesh_terms: D000328:Adult; D001667:Binding, Competitive; D001774:Blood Chemical Analysis; D002112:Calcifediol / Q000097:blood* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007231:Infant, Newborn; D007536:Isomerism; D011485:Protein Binding; D014809:Vitamin D-Binding Protein / Q000378:metabolism*
chemical_list: D014809:Vitamin D-Binding Protein; D002112:Calcifediol